In Vivo Administration of Anti-HIV Hyper-Immune Eggs Induces Anti-Anti-Idiotypic

bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

In vivo administration of anti-HIV hyper-immune eggs induces anti-anti-idiotypic

antibodies against HIV gp120 peptides (fragments 308-331 and 421-438) and against HIV

gp41 peptide (fragment 579-601) which have demonstrable protective capacity.

Angel Justiz-Vaillant1*, Belkis Ferrer-Cosme2, Monica Fisher Smikle3, Oliver Pérez4 1Department of Para-Clinical Sciences. Faculty of Medical Sciences. The University of the West Indies. St. Augustine. Trinidad and Tobago. West Indies. 2Higher Institute of Medical Sciences of Santiago de Cuba. Cuba. 3Department of Microbiology. University Hospital of the West Indies. Mona Campus, Kingston, Jamaica. 4Department of Immunology, Instituto de Ciencias Básicas y Preclinicas “Victoria de Girón”. Cuba. *Corresponding author: Dr. Angel Justiz Vaillant [email protected]

Abstract

Isolation of antibodies from the egg yolk of chickens is of particular interest as a source of

specific antibodies for oral administration to prevent infections and use them as

immunodiagnostic reagents. The use of birds in antibody production results in a reduction in the

use of laboratory animals. Immunized chickens produce larger quantities of antibodies (2000 mg

IgY/month) than rodents (200 mg IgG/month) in the laboratory. According to Jerne's network

theory, it is possible to produce an antibody against the antigen-binding site of another antibody.

This study assessed the hypothesis that immunization with viral peptides (immunogens) could

provide a potent immune response that could be evaluated in chicken eggs. Human

immunodeficiency virus 1(HIV-1) is used as an immunogen. The second hypothesis was that an

orally administered antibody stimulates the production of a complementary antibody, the so-

called anti-idiotypic antibody, which can potentially be therapeutical. This study reports and bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

analyzes the use of eggs as therapeutic agents. We wanted to test the hypothesis that feeding

chicks with hyperimmune eggs stimulates the production of anti-anti-idiotypic antibodies that

neutralize the original HIV antigen fragments 308-331 or 421-438 of gp120 or fragment 579-601

of gp41. Future research could entail an anti-idiotype strategy for prophylactic vaccines. It is

vital to note that it may need an anti-idiotype response to prime immunity against an HIV viral

epitope, which may be used as a secondary element. The use of anti-idiotype immune responses

in infected individuals may shift the balance of the immune system, allowing the organism to

manage HIV infection. Therefore, it may be an avenue for immunotherapy to improve the fight

against HIV infections. However, more studies and clinical trials are required to demonstrate

similar human immune responses as observed in birds.

Keywords: Microbiology, Vaccinology, Anti-idiotypic antibodies, ELISA, Human

Immunodeficiency virus

Introduction

The species often chosen for antisera production are rabbits or other mammals. There has been a

growth in the use of hens for antibody production. Immunoglobulin (Ig)-Y is the primary

antibody produced by birds and has advantages over mammalian IgG. There is no activation of

the mammalian complement system, no cross-reactivity with human anti-mouse antibody

(HAMA), rheumatoid factors, or human blood group antigens (lack of heteroagglutinins) [1]. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

According to Jerne's network theory, antibodies contain in their variable region a representation

of the 'universe' of antigenic structures, the idiotype. It is possible to produce antibodies against

antigen-binding sites of antibodies directed to the antigen [2,3]. The production of a humoral and

mucosal immune response in rabbits fed daily doses of the MOPC-315 murine IgA antibody,

supporting the hypothesis that human exposure to xenogeneic antibodies, most commonly bovine

milk immunoglobulins, may induce the production of anti-idiotypic antibodies [4].

We wanted to support the same hypothesis by demonstrating the animals' capacity to orally feed

hyperimmune eggs to induce systemic immune responses against the same idiotype. The study's

first set was to show that chickens immunized with the HIVgp120 peptide produced specific

anti-HIV antibodies. The second set of studies demonstrated that cats fed anti-HIV hyper-

immune eggs could develop antibodies that can inhibit egg yolk anti-HIV antibody binding to the

HIV gp120 (fragment 254-274) peptide (original antigen), showing that the anti-HIV antibody

raised in cats after feeding was anti-anti-idiotypic [3].

The main goal of this study was to raise chicken antibodies against a crucial public health

microorganism, human immunodeficiency virus 1. Immunogens were prepared using this

microorganism. The chickens were vaccinated intramuscularly. After booster immunization,

most eggs were collected and assessed for the presence of specific antibodies. The most

important result was the production of many anti-HIV antibodies in chicken eggs, with a high

quantity of antibodies that inhibit the binding of Ab1 antibodies to the original antigen. Enzyme-

linked immunosorbent assays were used to detect the presence of anti-HIV antibodies. bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Materials and methods

Ethical approval was granted by the Campus Ethics Committee of the University of the West

Indies, Mona Campus. The laboratory work described was conducted under EU Directive

2010/63/EU for experiments.

A higher confidence level indicates a larger sample size. Power is the probability of statistically

significant evidence of disparity among the groups, given the population's variation. A higher

power requires a larger sample size. A previously published HIV vaccine study included a small

number of animals [4]. This new experiment documented here aims to describe earlier findings

of the development of specific antibodies using oral hyperimmune eggs as feeding. This was a

pilot study of an oral vaccine against HIV, using cat-fed chicken eggs as a therapeutic option

[4].

The chicken house is made of wood and recreates the natural habitat of chickens. It has a black

shade around the house that provides 70% shade. The area to the sides and front of the chicken

house was weeded and cleaned for esthetics to minimize bacterial growth. The shaded house

helped reduce the admission of sun rays, which, coupled with the zinc roof, would increase the

chicken house's temperature, which is undesirable for the birds. The chickens were housed in

separate cages. Egg collection was performed daily, including on weekends.

The chicken house material consisted of a Hypo layer feed, 1.5m gauged mesh, 0.25 gauge

expanding metal, and push broom shower. Other elements include water buckets, bleach (sodium bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

hypochlorite), hydrogen peroxide (5%), egg trays, industrial gloves, sterile latex gloves, sterile

applicator sticks, 27G1/2 size syringe, feed tray, and water trays.

The chickens were tested and observed for four months or another specified period. No vitamins

or medications of any sort were administered to the birds under the project. The animal house

was washed out with cleaning products twice daily, and fecal content was removed in keeping

with a sound biosecurity practice. The chickens were watched daily for daily well-being and

health and supervised by a veterinary technician. Fresh water and corn were provided to birds

under free demand.

Immunogenicity studies of experimental HIV vaccine candidates in chickens

All grade reagents used in this study were commercially available. They were purchased from

Sigma-Aldrich. The study was repeated three times and similar results were obtained. The HIV

immunogens used in these experiments were keyhole limpet hemocyanin (KLH) conjugated to

the following HIV peptides:

1. Fragments 308-331 and 421-438 of the HIV gp120 [5]

2. Fragments 579-601 of the HIV gp 41 [5].

This peptide, derived from the V3 loop of HIV gp120, completely blocks the fusion inhibition

activity of antisera to recombinant human HIV proteins and inhibits the infection of primary

human T cells by HIV-1 as syncytium formation.

The second peptide, 421-438, belongs to the CD4 binding site of HIV gp120.

We also used fragments 579-601 from the N-terminal heptad repeat (NHR) and loop region

(loop) of HIV gp 41 (fragment 579-601) [2].

The amino acid sequences of HIV peptides and their references are cited below [5].

HIV gp 41 (fragment 579-601): Arg-lle-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-

Leu-Gly lle-Trp-Gly Cys-Ser-Gly Lys [6].

HIV gp120 (308-331): Asn-Asn-Thr-Arg-Lys-Ser-lle-Arg-lle-Gln-Arg-Gly-Pro-Gly-Arg-Ala-

Phe-Val-Thr-lle- Gly-Lys-lle-Gly [7].

HIV gp120 (421-438): Lys-Gln-Phe-lle-Asn-Met-Trp-Gln-Glu-Val-Gly-Lys-Ala-Met-Tyr-Ala-

Pro-Pro [8].

Dimerization of HIV peptides and preparation of HIV immunogens

The C-terminal cysteine was added to the amino acid sequences of HIV peptides (fragment 579-

601 of HIV gp 41 and fragments 308-331 and 421-438 of HIV gp120. These peptide fragments

are dimerized via cysteine oxidation in dimethyl sulfoxide [9]. Each HIV peptide was dissolved

in 5% acetic acid to a final concentration of 5.1 mg/mL. The pH of the medium was adjusted to 6

with 1 M (NH4)2CO3, and dimethyl sulfoxide was added to 20% of the final volume, and after

four h at room temperature (RT), the solute was extracted. Subsequently, the peptide was

dissolved in 3 mL of 5% trifluoroacetic acid and precipitated with 35 mL of cold ether. The

precipitate was dialyzed against 1.2 L of deionized water, pH 7 at 4°C overnight [2].

Then, 1 mg of keyhole limpet hemocyanin (KLH) was diluted in 2.1 ml 0.1 M borate buffer

(1.24 g boric acid, 1.90 g sodium tetraborate, pH 10, in 500 mL deionized water). In a 20 ml

glass tube, with gentle stirring, 1.1 µmol of each HIV synthetic peptide (with C-terminal cysteine

added) and 0.22 milliliters 0.3% glutaraldehyde solution (ACS reagent grade, pH 5.5, Sigma-

Aldrich) at RT were slowly mixed and left to stand for 1.50 hrs. A yellow coloration was

observed, indicating that the conjugation process was successful. To blocking the excess of

glutaraldehyde, 0.26 ml of 1 M glycine (Sigma-Aldrich) was added to block the excess

glutaraldehyde, and the mixture was left for 32 min at RT. Each HIV-hemocyanin conjugate was

then dialyzed against 1.3 liters 0.1 M of borate buffer, pH 8.4 through the night at 4°C. The same

buffer was then used to dialyze the preparations for 8 h at 4°C. The dialysates were stored at 4°C

until further use. 2.

Chicken immunization

Nine healthy brown Leghorn layer hens (three per HIV immunogen), aged seven months, were

immunized intramuscularly (IM) at multiple sites on the breast with a specific KLH-conjugated

HIV peptide vaccine (KLH-conjugated HIV peptides were HIVgp41 fragment 579-601,

HIVgp120 fragment 308-331, or HIVgp120 fragment 421-438). Chickens were vaccinated on

day 0, with 0.5 mg/mL of the immunogens in 0.5 ml of complete Freund's adjuvant (Sigma-

Aldrich), and on days 14, 28, and 45 after the first immunization, hens received booster doses of

0.25 mg/mL of the immunogen in 0.5 mL incomplete Freund's adjuvant. Eggs were collected

daily before and after the immunization.

Additionally, nine healthy brown Leghorn chickens were fed hyper-immune eggs for 45 days

(three birds for each candidate vaccine: KLH conjugated to HIV peptides, HIVgp41 fragment

579-601, HIVgp120 fragment 308-331, or HIVgp120 fragment 421-438). Likewise, three

healthy brown Leghorn chickens were fed non-hyper-immune eggs for 45 days, and their

response to each peptide was measured. The procedure was blinded to the procedure. The

scientist did not intervene in the feeding of the birds nor knew none of the groups.

Feeding of chicks

Feeding of chicks with HIV hyper-immune eggs with a titer of 1:15000 was fed to 15) chicks

aged zero and divided into three groups fed for one month. The first group of five chicks was

supplied with hyper-immune eggs against fragment 579-601 of the HIV gp 41 (fragment 579-

601) peptide and corn. Likewise, the second group of five chicks was fed a hyper-immune egg

against segments 308-331 of the HIV gp120 peptide and corn. The third group of five chicks was

supplied with a hyper-immune egg against fragments 421-438 of the HIV gp120 peptide and

corn. An additional ten chicks were divided into two groups: a group of five chicks that received

non-hyperimmune HIV eggs and corn, and the last group of five chicks was fed with only corn.

The feeding schedule was a blinding up process, the scientist did not take part in this process,

and they did not know that each group was being treated. At the end of the feeding schedule,

blood samples were obtained from all the chicks and investigated using an inhibition ELISA [5].

The water-soluble fraction (WSF) contains an elevated IgY concentration, which is separated

from the lipid content by the partial application of the Polson methodology [9], which uses only

chloroform, as follows: Despues de : van minúsculas y termina con ; y ultimo se une con and bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1. wash the egg with warm water and crack the eggshell carefully;

2. The egg yolk was manually separated from the egg white, and the egg content was placed

on tissue paper to remove as much egg white as possible.

3. The egg yolk was washed four times with 100 mL of PBS (pH 7.4) (Sigma-Aldrich).

4. break the egg yolk membrane and pour it into a 50 mL tube. The egg yolk was diluted 1:5

in PBS (pH 7.4), and an equal volume of chloroform (ACS reagent grade, Sigma-

Aldrich) was added.

5. The mixture was shaken and vortexed, and the tube containing the mixture was rolled on

a rolling mixer for 15 min.

6. centrifuge the mixture for 15 min (2000×g at 18°C); and

7. decant the supernatant, which contains the diluted egg yolk plasma WSF rich in IgY (25

mg/mL), which is approximately four times the IgY concentration present in the serum (6

mg/mL).

Indirect enzyme-linked immunosorbent assay

Preparation of ELISA reagents

The coating buffer was prepared by adding 3.7 g Sodium Bicarbonate (NaHCO3) and 0.64 g

Sodium Carbonate (Na2CO3) in 1 L of distilled water. Phosphate buffered-saline Tween-20

(10% PBS-Tween 20, pH 7.2) was made by dissolving the following reagents 0.2 g of KCl, 8 g

of NaCl, 1.45 g of Na2HPO4, 0.25 g of KH2PO4, and 2 mL of tween-20 in 800 mL of distilled

water. The pH was adjusted to 7.2, and distilled water was added to adjust the volume to 1 L,

which was then sterilized by autoclaving. The blocking solution was prepared by mixing 0.1 g

KCl, 0.1 g K3PO4, 1.16 g Na2HPO4, and 4.0 g NaCl in 500 mL distilled water, pH 7.4. Fifteen

grams of non-fat dry milk was added to complete the preparation of this solution, and 15 g of bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

non-fat dry milk was added. The sample/conjugate diluent was prepared by adding 15 g of non-

fat dry milk and 2.5 mL of 10% Tween 20 to 500 mL of PBS [5].

ELISA for anti-HIV peptide antibodies

The 96-well polystyrene microplates (U-shaped bottom, Sigma-Aldrich) were coated with 100

ng of fragment 579-601 of HIV gp 41, fragments 308-331, or fragment 421-438 from HIV gp120

in coating buffer for 1 h at 37°C. Each microplate was washed four times with 10% PBS-Tween

20, and the blocking solution (3% non-fat milk in PBS) was added to each well (51 µL). The

microplates were incubated for 1.30 h at RT. The microplates were washed as previously

described. Fifty microliters of WSF diluted 1:50 with the sample diluent was added to the wells.

Each microplate was then incubated for 1 h at RT and washed four times, as previously

described. Then, 50 µL of horseradish peroxidase-labeled anti-IgY conjugate (Sigma-Aldrich)

diluted 1:30,000 was poured into each well. The microtiter plates were incubated again for 1h at

RT and washed four times. A volume of 50 µL tetramethylbenzidine (TMB, Sigma-Aldrich) was

added. After further incubation for 16 min in the dark, the reaction was stopped with a solution

of 3M HCl, and each microplate was read in a microplate reader at 450 nm. The cut-off value

was assessed based on the mean optical density (OD) of the negative control time of 2 [2-5]. The

cut-off points of ELISAs for the detection of the anti-HIV peptide (579-601), anti-HIV peptide

(308-331), and anti-HIV peptide (421-438) were 0.42, 0.40, and 0.44, respectively.

Positive and negative controls were homemade. Four positive controls were used in each assay,

prepared from egg yolk samples with the highest titers of specific anti-HIV peptide antibodies, bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

and their OD values were between 1.20 and 1.50 at 450 nm. Four negative controls were used in

each assay. They were prepared from the egg yolks of non-immunized animals, and they showed

OD values of 0.170-0.20 at 450 nm. Three replicates of each WSF sample per bird, collected on

day 60, were assayed for the presence of anti-HIV peptide antibodies using ELISA.

Inhibition immunoassays

Ninety-six-well polystyrene microplates (U-shaped bottom, Sigma-Aldrich Co, St Louis

Missouri) were coated with 50 µL/well of 1 ng/µL solution of fragments 308-331 or 421-438

from HIV gp120 peptides or fragment 579-601 of HIV gp41 (Sigma-Aldrich) in carbonate-

bicarbonate buffer (pH 9.6; Sigma-Aldrich) for 4 h at 37°C. The microplates were then washed

four times with PBS Tween-20 and blocked with 3% non-fat milk in PBS, 25 µL/well, 1h, RT).

Triplicates of serial doubling dilutions of chick sera diluted in PBS 3% non-fat milk (pH 7.4)

were added to the microplate, which was then incubated for 90 min at 37°C. The microplates

were washed four times with 150 µL of PBS-Tween 20, and 25 µL of a WSF with anti-HIV

gp120 (fragments 308-331 or 421-438) antibody titer of 1:5000 or anti-HIV gp41 (fragments

579-601) with an antibody titer of 1:1000 were added to each well. Following incubation for 90

min at 37°C, the microplates were washed 4X and 25 µL of a 1:30,000 dilution of rabbit anti-

chicken IgY-HRP was added to each well. The microplates were then incubated at 37°C for 1 h

before final washing (PBS-Tween 20, 4 times), and 25 µL of TMB was added before incubation

in the dark for 15 min. The reaction was stopped using 3M H2SO4. Microplates were read at 450

nm wavelength using a microplate reader. The percentage inhibition (%I) was calculated using

the following formula: The intra-assay coefficient of variation was then calculated.

XOD of sample-XOD of blanks %I=100- ______x100 XOD 100% HIVgp120 or HIVgp41 peptide binding to anti-gp120 or anti-gp41- XOD of blanks

Results and Discussion Table 1 shows the water-soluble fractions (WSF) of egg yolks collected at zero and 60-days

post-immunization were assayed for the presence of anti-HIV peptide antibodies using ELISA

(six samples in total were assayed and evaluated three times). These immunogenicity results

showed that HIV peptide vaccines effectively produced strong anti-HIV immune responses in

immunized brown Leghorn layer hens. There was a statistically significant difference (P<0.01)

that explains the notable degree of difference (related to the anti-HIV antibody levels in the egg

yolks) between pre-immunized and post-immunized birds in the three experimental vaccines [2].

These immunogenicity results showed that HIV peptide vaccines effectively produced a strong

anti-HIV immune response in immunized brown Leghorn layer hens.

Table 1. Results of immunogenicity studies of experimental HIV vaccine in brown Leghorn layer hens Mean optical density (XOD), Pre- (day 0) and post-immunization (d 45) titers, SD, standard deviation. XOD (SD), day 0 (Pre- XOD (SD), 45 days Candidate vaccines P-value immunized birds) post-immunized birds HIV gp 41 (fragment 0.170 (0.021) 0.885 (0.044) <0.001 579-601) 3 birds HIV gp120 (fragment 0.156 (0.015) 0.910 (0.023) <0.001 308-331) 3 birds HIV gp120 (fragment 0.188 (0.01) 0.865 (0.037) <0.001 421-438) 3 birds

The results showed nine healthy brown Leghorn layer hens (three birds per HIV candidate

vaccine), aged seven months, that were immunized IM at multiple sites on the breast with three bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

specific KLH-conjugated HIV peptide vaccines (five birds per candidate) on days 0, 15, 30, and

45. Eggs from the pre-immunized and post-immunized birds were collected. The WSF of each egg

[9] was evaluated using an ELISA kit. The coefficient of variation was less than 10%. This

suggests that the assays are reproducible ( Supporting Information). We could not compare our

HIV tests with a commercially prepared assay because such a study was not available at the time

of this study, and it is still not known.

Table 2. Results of immunogenicity studies of experimental HIV vaccine in brown Leghorn layer hens Mean optical density (XOD) and standard deviation of birds fed or not hyper- immune egg for 45 days. XOD (SD), day 45 (Birds XOD (SD), 45 days (Birds Candidate fed with non-hyper- fed with hyper-immune P-value vaccines immune eggs) eggs) HIV gp 41 0.250 (0.023) 0.995 (0.068) 0.001 (fragment 579-601) HIV gp120 0.312 (0.017) 0.859 (0.040) <0.001 (fragment 308-331) HIV gp120 0.278 (0.012) 0.773 (0.021) <0.001 (fragment 421-438)

Table 2 shows a significant statistical significance between birds fed non-hyperimmune eggs and

birds fed hyperimmune eggs 45 days post-immunization. This corroborates that hyper-immune

eggs cause oral immunization and can be considered an anti-idiotypic vaccine.

Table 3. Inhibition experiments. Mean of binding inhibition percentage (X%I), and Standard deviation (SD).

X%I (SD) of fed chicks X%I (SD) of non-fed Candidate with hyper-immune chicks with hyper-immune p-value vaccines eggs and corn (15-days eggs and corn post-immunization) HIV gp 41 (fragment 3.13 (0.37) 18.05 (2.40) 0.007 579-601) bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

HIV gp120 (fragment 2.41 (0.63) 19.62 (3.13) 0.009 308-331) HIV gp120 (fragment 3.19 (0.51) 13.92 (3.96) 0.041 421-438)

Table 3 shows the results of the inhibition of fragments of HIV gp120 (fragments 308-331 or

421-438) or HIV gp41 (fragment 579-601) and anti-HIV gp120 or anti-HIV gp41 reactions by

anti-anti-idiotypic HIV gp120 (fragments 308-331 or 421-438) or HIV-gp41 antibodies,

respectively. This inhibition was statistically significant (P <0.05).

The binding inhibition assay showed that 13.9%–20.3% inhibition of the binding of avian anti-

HIV gp 41 (fragment 579-601) or anti-HIV-gp120 antibodies (Ab-1) to immobilized HIVgp41,

HIVgp120 peptide by anti-HIV gp 41 (fragment 579-601) or anti-HIV gp120 (fragments 308-

331 or 421-438) antibodies present in serum sample replicates of chicks tested, suggesting that

the chicks anti-HIV gp 41 (fragment 579-601) or anti-HIV-gp120 antibodies (fragments 308-331

or 421-438) were anti-anti-idiotypic antibodies. This inhibition was not observed in the sera of

chicks that were not fed with hyperimmune eggs. This confirms the hypothesis that feeding

chicks with hyperimmune eggs stimulates the production of anti-anti-idiotypic antibodies that

neutralize the original HIV antigen (gp120 or gp41).

Table 4. Inhibition of fragments of HIV gp120 (fragments 308-331 or 421-438) or HIV gp41 (fragment 579-601) and anti-HIV gp120 (fragments 308-331 or 421-438) or anti-HIV gp41 (fragment 579-601) reaction by anti-anti-idiotypic HIV gp120 (fragments 308-331 or 421- 438) or HIV-gp41 antibodies, respectively. Mean of binding inhibition percentage (X%I), and Standard deviation (SD).

X%I (SD) of non-fed X%I (SD) of fed chicks chicks with hyper- with hyper-immune eggs Candidate vaccines p-value immune eggs (given corn and corn (30-days post- and water) immunization) HIV gp 41 (fragment 3.13 (0.37) 20.08 (3.23) 0.011 579-601) 3 birds bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

HIV gp120 (fragment 2.41 (0.63) 18.55 (2.47) 0.005 308-331) 3 birds HIV gp120 (fragment 3.19 (0.51) 16.79 (3.85) 0.024 421-438) 3 birds

Table 4 shows that chicks fed with corn and water failed to inhibit the binding or reactivity of

fragments of HIV gp120 (fragments 308-331 or 421-438) or HIV gp41 (fragment 579-601) and

anti-HIV gp120 (fragments 308-331 or 421-438) or anti-HIV gp41 (fragment 579-601) by anti-

anti-idiotypic HIV gp120 (fragments 308-331 or 421-438) or HIV-gp41 antibodies, respectively;

and again the chicks fed with hyper-immune eggs inhibited successfully the reactivity,

suggesting that the anti-anti-HIV antibody against each fragment of gp120 or gp41 was able to

recognized the original antigen and therefore reacting and protecting against it.

Table 5. Inhibition of fragments of HIV gp120 (fragments 308-331 or 421-438) or HIV gp41 (fragment 579-601) and anti-HIV gp120 (fragments 308-331 or 421-438) or anti-HIV gp41 (fragment 579-601) reaction by anti-anti-idiotypic HIV gp120 (fragments 308-331 or 421- 438) or HIV-gp41 antibodies, respectively. Mean of binding inhibition percentage (X%I), and Standard deviation (SD).

X%I (SD) of fed X%I (SD) of fed chicks with hyper- chicks with hyper- Candidate immune eggs (15- immune eggs (30- t-value p-value vaccines days post- days post- immunization) immunization) HIV gp 41 (fragment 18.05 (2.40) 20.08 (3.23) -1.24 24.71% 579-601) HIV gp120 (fragment 19.62 (3.13) 18.55 (2.47) 0.66 52.66% 308-331) HIV gp120 (fragment 13.92 (3.96) 16.79 (3.85) -1.27 23.19% 421-438)

These experiments did not reject the null hypothesis, indicating that there was no statistically

significant difference between the variables analyzed. Birds fed hyper-immune eggs 15 days or bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

30 days post-immunization were able to induce the same stimulated immune response against the

original antigen: HIV gp120 (fragments 308-331 or 421-438) or HIV gp41 (fragment 579-601).

It was shown that feeding chicks with anti-idiotypic HIV gp41 (fragment 579-601) or gp120

peptide antibodies induced anti-peptide antibodies (Ab-3), which also recognized a recombinant

HIV gp41 (fragment 579-601) or HIV gp120 (fragments 308-331 or 421-438), and also inhibited

its interaction with anti-HIV-gp41 or anti-HIV-gp120 antibodies (Ab1). This study provides

evidence that is supported by a previous study in which intramuscular immunization of BALB/c

mice with anti-idiotypic HIV gp160 peptide antibodies induced anti-peptide antibodies (Ab-3),

which also recognized a recombinant HIV gp160 preparation, and inhibited its interaction with

anti-HIV-gp160 antibodies (Ab1) [11]. Anti-idiotypic vaccines in HIV/AIDS, using a

monoclonal antibody (mAb 13B8.2) directed against the CDR3-homologous CD4/D1 region has

previously been reported in the literature [4,12]. Our results are also supported by a study on the

development of an idiotype-anti-idiotype network of antibodies to bovine serum albumin (BSA)

in eggs from chickens immunized with BSA [13]. It has also been reported that HIV-1 gp160-

specific secretory IgA was detected in the saliva of all rabbits orally immunized with HIV-

immunosomes, neutralizing HIV infectivity in vitro [14]. Our study showed a statistically

significant percentage inhibition. Oral immunizations had a p-value < 0.05, which indicated the

presence of anti-anti-idiotypic antibodies (Ab3) against HIV gp120 (fragments 308-331 or 421-

438) and HIV gp 41 (fragment 579-601) fragments, which inhibited the formation of complexes

between HIV gp120 (fragments 308-331 or 421-438) peptide or HIV gp 41 (fragment 579-601)

and anti-HIVgp120 or anti-HIV gp 41 (fragment 579-601) idiotypic antibodies, respectively.

Boudet et al. (1992) reported that one of the well-characterized motifs mapped to a loop within

the third hypervariable region (V3) of the exterior envelope glycoprotein gp120 at amino acid

positions 308–331 and is referred to as the principal neutralizing determinant (PND). The

sequence of this V3 loop raises the immunogenicity and diversity of the antibody response to

PND. We show here that this neutralization-related motif is highly immunogenic in HIV-positive

subjects and experimentally immunized primates and rodents subjected to various anti-HIV

immunization regimens [15]. Our study showed that this peptide could generate a robust immune

response in chickens, consistent with previous research by Boudet et al. The immunogenicity

study was statistically significant (p <0.05), suggesting that it could be a good immunogen for a

candidate vaccine.

Bell et al. (1992) reported the use of a series of overlapping synthetic peptides derived from a

conserved region of the envelope gp41 (aa 572-613). They identified an immunodominant 12-

mer peptide sequence, gp41(8) (aa 593-604), which consistently elicited both T cell blastogenic

and antibody responses in asymptomatic HIV-seropositive individuals but not in ARC and AIDS

patients [16]. Linear regression analysis showed that in asymptomatic persons, there was a strong

positive correlation (P less than 0.0005) between the absolute CD8+ T cell and the magnitude of

blastogenic responses to gp41(8) (aa 593-604) [16]. These experiments suggest that the involved

peptides have a high immunogenicity capacity, as suggested by our study. Therefore, the

fragment HIV gp 41 (fragment 579-601) (572-613) is a promising vaccine candidate, as shown

in our study in immunogenicity studies and binding inhibition assays.

The first (B138) is linear and spans the envelope residues 421-438; the second (1005/45)

encompasses amino acids 418-445 and is cyclized by a disulfide bond joining its terminal

cysteines. Both species have been shown to inhibit syncytia formation in a conventional

bioassay, with B138 being the most efficient. Both peptides elicit high titers of anti-peptide

antibodies in immunized mice, rabbits, and goats, with titers exceeding 1:10 in many cases [8].

Our study does not show that anti-idiotypic antibodies inhibit syncytia formation, because this

was not our aim. We are not aware of the literature on other immunogenicity studies performed

with the HIV gp120 (fragments 308-331 or 421-438)peptide fragment 421-438. Our study makes

an original contribution to the literature.

According to Jerne’s network theory, antibodies have in their variable regions an image of the

‘universe’ of antigenic arrangements, the idiotype (Id). Therefore, antibodies can be recognized

as antigens by the immune system under normal circumstances, and these interactions are called

Id-anti-Id reactions, which can be used to manipulate the immune system [17].

Egg yolk is a critical and alternative source of polyclonal antibodies. Of course, they present

some advantages over mammalian antibodies in terms of animal welfare, productivity, and

specificity [1]. Moreover, because of its phylogenetic distance and structural molecular features,

IgY is more suitable for treatment and diagnostic purposes than mammals because of its lack of

reactivity with the human complement system and B cell repertoire [1]. However, IgY has a

greater avidity for conserved mammalian polypeptides [18,19]. The main antibody (IgY) present

in avian blood is transmitted to the offspring and stored in egg yolks, facilitating the non- bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

invasive harvesting of many antibodies [20,21]. Indeed, chicken IgY antibodies can be used

therapeutically and in rapid diagnostic tests [22-25].

Chicken IgY provides a more ethical and humane approach to therapeutic and diagnostic

methods that use these polyclonal antibodies. It has a lower cost of return than mammalian

antibodies [26], with the ability to produce more antibodies with fewer animals involved. It can

translate to lower prices for diagnostic tests or therapeutic methods that utilize chicken IgY.

Diagnostically, IgY does not cross-react with Fc receptors of rheumatoid factors, HAMA, human

blood group antigens, or activates the complement system. Thus, there are fewer false-positive

results when using IgY in immunological assays, making it more specific to detect several

antigen classes [1,26].

Therapeutically, IgY has the potential to be used in the treatment of many infections. These

include infectious diseases caused by Helicobacter pylori [27], enterotoxigenic E. coli,

and Salmonella spp. [26]. Other problems include acute and chronic pharyngitis [28] and

infantile rotavirus enteritis [29]. The prevention of oral colonization by Streptococcus

mutans [30] and the potential for greater immunotherapy (such as for HIV) make IgY critical in

modern medical treatment. Due to IgY properties, less toxicity and side effects, and fewer

allergic reactions make it more suitable than previously considered immunotherapies involving

mammalian antibodies. Its therapeutic potential makes it a necessary treatment that requires

more consideration, especially with bacterial resistance to existing antibiotics and the emergence

of novel strains of viruses [31]. Egg yolk antibodies were also obtained. Egg white

immunoglobulin with specificity to staphylococcal protein A was detected in the immunized

hens. It inhibits S. aureus growth in vitro [32]. A sandwich ELISA showed anti-SpA antibodies bioRxiv preprint doi: https://doi.org/10.1101/2021.05.02.442298; this version posted May 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

in hens immunized with titers of at least 5-folds compared to pre-immunized hens ten days post-

immunization [32]. Therefore, the whole egg could be considered as a drug to combat

microorganisms. In vitro experiments support the hypothesis that the idiotypic-anti-idiotypic

network plays a role in immunoprotection, which deserves further study. The authors suggest

that eggs from chickens immunized with specific immunogens may be considered a special type

of oral anti-idiotypic vaccine. It was shown by development of anti-anti-idiotypic antibodies that

were developed against various epitopes [33].

Conclusions

Future research could entail an anti-idiotype strategy for prophylactic vaccines. It is vital to note

that it may need an anti-idiotype response to prime immunity against an HIV viral epitope, which

may be used as a secondary element. The use of anti-idiotype immune responses in infected

individuals may shift the balance of the immune system, allowing the organism to manage HIV

infection. We hypothesized that feeding chicks with hyperimmune eggs would stimulate the

production of anti-anti-idiotypic antibodies that neutralize the original HIV antigen (gp120 or

gp41). Therefore, it may be an avenue for immunotherapy to improve the fight against HIV

infections. However, more studies and clinical trials are required to demonstrate similar human

immune responses as observed in birds.

Conflict of Interest: The authors declare that conflict of interest does not exist.

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